In order to perform their cellular roles, proteins must fold into complex three-dimensional structures. Protein folding is an important basic scientific question, which also has significant medical implications due to the prevalence of devastating protein folding diseases. Many neurodegenerative diseases, such as Alzheimer's, Huntington's, Parkinson's disease and ALS (Lou Gehrig's disease) are caused by the misfolding and aggregation of proteins. Although a few cellular proteins are capable of achieving their correct three- dimensional native structure on their own, most proteins in the cell require the assistance of proteins called molecular chaperones, which aid in the folding process. The goal of this research is to understand how protein folding occurs in the cell. This research will focus on how cotranslational chaperones aid in the folding of newly translated proteins as they emerge from the ribosome. The objective of this proposal is to understand the mechanisms by which cotranslational chaperones fold newly synthesized proteins. Specifically, we have identified the following critical questions that will allow us to understand the mechanisms governing this process: which chaperones interact with which nascent chains, when do different chaperones initiate these interactions and what is the interplay between different cotranslational chaperones, and how does nutrient availability affect the function of this folding network? Our strategy will be to use a combination of in vivo genomic and functional assays to obtain functional insights into the roles of different cotranslational chaperones in folding of the cellular proteome. The first specific aim will use genomic ribosome-associated chaperone enrichment analysis to determine the subset of nascent chains that each cotranslational chaperone interacts with during translation. This approach will be coupled with functional assays of protein aggregation and ubiquitination to determine if these specific chaperone/substrate interactions are strictly required for efficient protein folding. In the second specific aim, a combination of genomic ribosome-associated chaperone enrichment analysis in different genetic backgrounds, lacking specific chaperone systems, and selective ribosomal profiling will be used to determine the overall architecture of the cotranslational chaperone network. Finally, the third specific aim will examine how the physiologically relevant effects of nutrient availability affect the interplay between the translational machinery, chaperones and their substrates. Successful realization of these aims will provide a detailed understanding of how the cotranslational chaperone network functions. A detailed, molecular understanding of this network should open up many avenues to design therapeutics to combat the underlying causes of devastating protein folding diseases.